The functional identification, biochemical purification and cloning of a novel cytosolic serine protease, QPP (Quiescent Proline di-Peptidase), which prevents quiescent lymphocytes from undergoing programmed cell death (PCD) forms the basis for the working hypothesis that apoptosis is blocked in resting lymphocytes by an active mechanism. QPP seems to be essential for the survival of resting T and B cells, because specific inhibition of this enzyme leads to activation of cellular caspases and PCD. The goals of this proposal are to substantiate these intriguing observations and to define the apoptosis pathway in G/o lymphocytes. I. to directly analyze the role of QPP in lymphocyte, a dominant negative (DN) variant will be constructed by mutating its catalytic site(s) such that it still binds its specific substrate, but no longer cleaves it. Wild type and DN QPP constructs will be expressed in vitro in cell lines and primary T cells, as well as in vivo by the use of the RAG-2 blastocyst ES complementation system. II. To understand the functional significance of QPP in the protection of lymphocytes from PCD, its physiological substrate(s) has to be identified. Two approaches will be used: i) rDN QPP protein as affinity matrix to extract the substrate from lymphocyte lysate; and ii) the yeast-two hybrid system with the DN QPP as bait for screening a lymphocyte cDNA library. The proteins identified by these methods will be verified as substrates of QPP by their susceptibility to cleavage by wild type enzyme, followed by N- terminal sequence analysis. III. Preliminary data indicate that the caspase cascade initiated in quiescent lymphocytes by blocking QPP differs significantly from other well characterized apoptotic pathways in these cells, such as irradiation- or Fas-induced PCD. Radioactively labeled zVADfmk, an irreversible caspase inhibitor, will be used as an active site-directed affinity reagent, in conjunction with 2D gel analysis, to identify the caspase(s) involved. IV. All cells tested so far contain a protease activity that resembles QPP in its blots, namely, a band of 1.7 kB that is expressed in all tissues and a band of 2.5 kB that is seen mainly in lymphoid cells and pancreas. Furthermore, the sequencing of QPP ESTs revealed clones that contain internal sequence gaps. Thus, it will be determined whether this enzyme is differentially spliced and/of differentially modified in a tissue specific manner. Taken together, these studies will provide new insights into the maintenance of homeostasis of resting lymphocytes in the immune system.